去离子甲酰胺在脑腱黄瘤病基因诊断中的应用  

Application of deionized formamide in genetic diagnosis of cerebrotendinous xanthomatosis

作　　者：宫磊[1] 张志坚 杨建课[1] 高继光[1] 戚之琳[2] Gong Lei;Zhang Zhijian;Yang Jianke;Gao Jiguang;Qi Zhilin(Department of Medical Biology,Wannan Medical College,Wuhu 241002,Anhui,China;Department of Medical Biochemistry,Wannan Medical College,Wuhu 241002,Anhui,China)

机构地区：[1]皖南医学院医学生物学教研室,安徽芜湖241002 [2]皖南医学院医学生物化学教研室,安徽芜湖241002

出　　处：《右江民族医学院学报》2021年第4期481-484,共4页Journal of Youjiang Medical University for Nationalities

摘　　要：目的探讨去离子甲酰胺在脑腱黄瘤病基因诊断中的应用价值,同时为高GC含量DNA片段的扩增提供方法上的参考。方法在反应体系中不含去离子甲酰胺以及去离子甲酰胺浓度为1%-10%的情况下采用热启动PCR扩增CYP27A1基因4个高GC含量的片段,然后进行琼脂糖凝胶电泳,再用Image J软件分析不同去离子甲酰胺浓度下目标条带的光强度值,比较差异,最后挑选特异性扩增产物测序分析。结果在不含去离子甲酰胺和去离子甲酰胺浓度为1%-6%的情况下,均扩增出目标片段;在不含去离子甲酰胺时,4个目标片段的扩增产物均存在非特异性带,而当去离子甲酰胺的浓度为4%-6%时,非特异性带消失。不同浓度(0-6%)条件下目标带的光强度值差异无统计学意义。特异性扩增产物经测序验证为目标片段。结论采用热启动PCR,同时向反应体系中添加去离子甲酰胺能有效扩增出高GC含量的DNA片段,并避免非特异性带的产生,但反应体系中去离子甲酰胺的浓度宜控制在4%-6%。这一方法不仅适用于脑腱黄瘤病的致病基因—CYP27A1基因的检测,也可用于其他富含GC的基因的检测。Objective To explore the application value of deionized formamide in genetic diagnosis of cerebrotendinous xanthomatosis,so as to provide methodological reference for the amplification of DNA fragments with high GC content.Methods Four segments of the CYP27A1 gene with high GC content were amplified by hot-start PCR in the reaction system without deionized formamide or with 1%-10%deionized formamide.Then agarose gel electrophoresis was performed on these segments.The software Image J was then used to analyze the light intensity values of the target bands at different concentrations of deionized formamide,and the value differences were compared.Finally,specific amplification products were selected for sequencing analysis.Results The target fragments were amplified when there was no deionized formamide as well as when there was deionized formamide at the concentration of 1%-6%.In the absence of deionized formamide,all the amplificated products of the four target fragments had non-specific bands.When the concentration of deionized formamide was 4%-6%,the non-specific band disappeared.There was no significant difference in the light intensity of the target zone at different concentrations(0-6%).The specific amplificated product was confirmed as the target fragment by sequencing.Conclusion Using hot-start PCR and adding deionized formamide to the reaction system can effectively amplify DNA fragments with high GC content and avoid the generation of non-specific bands.However,the concentration of deionized formamide in the reaction system should be restrained at 4%-6%.This method is not only applicable for the detection of CYP27A1(the pathogenic gene of cerebrotendinous xanthomatosis),but also can be used for the detection of other GC-rich genes.

关 键 词：去离子甲酰胺 热启动PCR CYP27A1基因 黄瘤病 脑腱性 

分 类 号：R589.2[医药卫生—内分泌]